Integrated temporal profiling of iPSCs-derived motor neurons from ALS patients carrying C9orf72, FUS, TARDBP, and SOD1 mutations
Guoming MaCongcong XiaBoyu LyuJie LiuFang LuoMingfeng GuanJunying WangLi SunZhang LinYan ChenYingwei MaoG. YuWenyuan Wang
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Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease that damages motor neurons in the central nervous system, causing progressive muscle weakness that ultimately leads to death. However, its underlying mechanisms still need to be fully understood, particularly the heterogeneity and similarity between various gene mutants during disease progression. In this study, we conducted temporal RNA-seq profiling in human induced pluripotent stem cells (hiPSCs) and iPSC-derived motor neurons (iMNs) carrying the C9orf72, FUS, TARDBP, and SOD1 mutations from both ALS patients and healthy individuals. We discovered dysregulated gene expression and alternative splicing (AS) throughout iMN development and maturation, and ALS iMNs display enrichment of cytoskeletal defects and synaptic alterations from premature stage to mature iMNs. Our findings indicate that synaptic gene dysfunction is the common molecular hallmark of fALS, which might result in neuronal susceptibility and progressive motor neuron degeneration. Analysis of upstream splicing factors revealed that differentially expressed RNA-binding proteins (RBPs) in ALS iMNs may cause abnormal AS events, suggesting the importance of studying RBP defects in ALS research. Overall, our research provides a comprehensive and valuable resource for gaining insights into the shared mechanisms of ALS pathogenesis during motor neuron development and maturation in iMN models.Keywords:
TARDBP
C9ORF72
Profiling (computer programming)
Background Expanded GGGGCC hexanucleotide repeats in the promoter of the C9ORF72 gene have recently been identified in frontotemporal dementia (FTD), Amyotrophic Lateral Sclerosis (ALS) and ALS-FTD and appear as the most common genetic cause of familial (FALS) and sporadic (SALS) forms of ALS. Methods We searched for the C9ORF72 repeat expansion in 950 French ALS patients (225 FALS and 725 SALS) and 580 control subjects and performed genotype-phenotype correlations. Results The repeat expansion was present in 46% of FALS, 8% of SALS and 0% of controls. Phenotype comparisons were made between FALS patients with expanded C9ORF72 repeats and patients carrying another ALS-related gene ( SOD1, TARDBP, FUS ) or a yet unidentified genetic defect. SALS patients with and without C9ORF72 repeat expansions were also compared. The C9ORF72 group presented more frequent bulbar onset both in FALS (p<0.0001 vs SOD1 , p=0.002 vs TARDBP , p=0.011 vs FUS , p=0.0153 vs other FALS) and SALS (p=0.047). FALS patients with C9ORF72 expansions had more frequent association with FTD than the other FALS patients (p<0.0001 vs SOD1 , p=0.04 vs TARDBP , p=0.004 vs FUS , p=0.03 vs other FALS). C9ORF72 -linked FALS patients presented an older age of onset than SOD1 (p=0.0139) or FUS mutation (p<0.0001) carriers. Disease duration was shorter for C9ORF72 expansion carriers than for SOD1 (p<0.0001) and TARDBP (p=0.0242) carriers, other FALS (p<0.0001) and C9ORF72 -negative SALS (p=0.0006). Conclusions Our results confirm the major role of expanded repeats in C9ORF72 as causative for ALS and provide evidence for specific phenotypic aspects compared to patients with other ALS-related genes.
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Amyotrophic lateral sclerosis (ALS) is the most common of the Motor Neurone Diseases and is commonly called Lou Gehrig's disease in North America. ALS is a rapidly progressive disease that attacks both upper and lower motor neurons responsible for controlling voluntary muscles, consequently leading to muscular paralysis and invariably death, most often within three years of diagnosis. The prevalence of ALS internationally is estimated to be between 3 – 7 people per 100,000, but the rapid progression and poor prognosis of the disease result in lifetime risk of around 1:300–1:400. Although the underlying cause of sporadic ALS remains a mystery, there is a rapidly growing list of genes in which mutations have been linked to inherited forms of familial ALS (fALS) which accounts for 5–10% of all ALS cases. The proportion of fALS kindreds that are linked to mutations in SOD1 depends on the geographical location and ranges from 13% in Australia to 20% in the USA and up to 50% in China. Behind C9ORF72 repeat expansions, mutations in SOD1 is by far the second leading known cause of ALS. Globally, there are over 150 disease-causing mutations in SOD1 associated with ALS. It is well established that the mutations cause a gain of function rather than loss of function, and the mutations have in common an ability to destabilize the native structure of the SOD1 protein [[1]Lindberg M.J. Tibell L. Oliveberg M. Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: decreased stability of the apo state.Proc Natl Acad Sci U S A. 2002; 99: 16607-16612Crossref PubMed Scopus (170) Google Scholar]. It is likely that much of the newly synthesized SOD1 never reaches its properly folded native state, since it is particularly supersaturated in the cellular milieu [[2]Ciryam P. et al.Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS.Proc Natl Acad Sci U S A. 2017; 114: E3935-E3943Crossref PubMed Scopus (54) Google Scholar] and that the disease-associated fibrillary aggregate structures it forms are thermodynamically favourable [[3]Rakhit R. Chakrabartty A. Structure, folding, and misfolding of Cu,Zn superoxide dismutase in amyotrophic lateral sclerosis.Biochim Biophys Acta. 2006; 1762: 1025-1037Crossref PubMed Scopus (140) Google Scholar]. In stark contrast, once it reaches its mature tertiary structure, it is amongst the most stable in the human body with a thermal melting point of around 90 C. The consequence of such destabilization are the SOD1 inclusions found in post mortem tissue, and downstream consequences such as ubiquitin-proteasome dysfunction, endoplasmic reticulum stress, mitochondrial dysfunction, and calcium dyshomeostasis leading to subsequent motor neuron death. The misfolding, and thus also the downstream consequences, can be propagated throughout the nervous system [[4]Crown A. et al.Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection.PLoS ONE. 2020; 15e0227655Crossref PubMed Scopus (8) Google Scholar]. Misfolded wild type SOD1 has been detected in sporadic ALS tissue [[5]Grad L.I. et al.Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.Proc Natl Acad Sci U S A. 2014; 111: 3620-3625Crossref PubMed Scopus (260) Google Scholar], of uncertain significance. It is likely that promoting the correct folding and maturation of SOD1 will address the underlying causes of SOD1 familial ALS, and perhaps will prove beneficial in sporadic ALS. Although limited to a handful of studies, previous attempts at developing SOD1 pharmacological chaperones have predominantly targeted the maintenance of the SOD1 dimeric state [[6]McAlary L. Yerbury J.J. Strategies to promote the maturation of ALS-associated SOD1 mutants: small molecules return to the fold.Neural Regen Res. 2019; 14: 1511-1512Crossref PubMed Scopus (4) Google Scholar] which is the starting point in misfolding from the native dimer. Whilst effective at preventing SOD1 aggregation of purified protein in vitro, the use of chemical crosslinkers as a therapeutic is limited by the fact that they typically have off-target effects such as toxic non-specific protein-protein crosslinking, and by the fact that much of newly synthesized protein in the cell never achieves the dimeric state. Previous work has shown that the selenium-based antioxidant compound ebselen pushes the folding equilibrium towards the native dimer in several SOD1-FALS mutants in vitro and in cells [[7]Capper M.J. et al.The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation.Nat Commun. 2018; 9: 1693Crossref PubMed Scopus (36) Google Scholar]. There is an important distinction between the dimer-promoting activity of ebselen and a mere tethering of already interacting subunits. In addition, the same work also demonstrated that ebselen could significantly increase the amount of disulfide oxidized SOD1 in cells [[7]Capper M.J. et al.The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation.Nat Commun. 2018; 9: 1693Crossref PubMed Scopus (36) Google Scholar]. Together these effects increase the thermal stability of the protein that should result in a protective effect in SOD1 ALS. It is at this point that the current work of Hasnain and colleagues [[8]Amporndanai K. et al.Novel Selenium-based compounds with therapeutic potential for SOD1-linked Amyotrophic Lateral Sclerosis.EBioMedicine. 2020; (In press)Summary Full Text Full Text PDF PubMed Scopus (4) Google Scholar] weighs in. Their paper, recently published in EBioMedicine, reports that feeding G93A SOD1 mice ebselen as part of their diet from 70 days old delayed disease onset by 10 days. The mice were fed food mixed with or without 0.016% w/w powdered ebselen at 24 mg/kg. They found that while ebselen significantly delayed the onset of ALS in mice, the treatment did not significantly alter mean survival time, body weight, or motor performance. While the ad libitum diet drug delivery method could be improved upon in future study, the in vivo drug efficacy is encouraging. To this end, the authors synthesized ebselen analogues and put them through a barrage of tests, from dimer dissociation, x-ray crystalization, and cell-based SOD1 toxicity experiments. Amongst the panel of analogues were a few that had enhanced activity when compared to ebselen. Future studies should determine the proportion of the drug that reaches the brain and the maximum tolerated dose of ebselen. It will be important to see if the lead analogues from the current work improve on the efficacy of ebselen in vivo. Importantly, ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use, enabling rapid translation to the clinic. Apart from the implication for future clinical practice, the study also demonstrates that pharmacological chaperones discovered through in vitro studies can translate to in vivo rodent studies and hopefully to humans with ALS. Dr. Yerbury declares no conflicts of interest. Dr. Cashman reports grants, personal fees and non-financial support from ProMIS Neurosciences, which does not conflict with this commentary. Drs. Yerbury and Cashman shared the writing and editing of this commentary. Novel Selenium-based compounds with therapeutic potential for SOD1-linked amyotrophic lateral sclerosisOur finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19). Full-Text PDF Open Access
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Superoxide dismutases(SOD)are known as biological cleaning agents of free radicals in vivo,and Cu,Zn-SOD also called SOD1,is one of the main forms.SOD1 mutations have been linked fatal human motor neuron diseases,such as amyotrophic lateral sclerosis,ALS.However,the precise roles of SOD1 in ALS occurenace and progression remianed unknown.This review will focus on the mechanism of SOD1's activation based on the structural conformation analysis of SOD1 and the copper chaperone for SOD1(CCS).Meanwhile,the latest research progresses of the possible pathogenic mechanisms in ALS will also be discussed.
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Amyotrophic lateral sclerosis(ALS)is a progressive neurodegenerative disorder.Mutations in the Cu,Zn-superoxide dismutase(SOD1)gene cause ALS by an unknown gain-of-function mechanism.Current researches show that diminishing SOD1 mutation enzymatic activity does not account for motor neuron lose in ALS,while direct toxicity of variant SOD1 protein or aggregation of SOD1 protein may impart cell function and cause motor neuron death.Though motor neuron death is selective,other cells also participate in this course.The author reviewed recent advances in the relationship between mutant SOD1 and Amyotrophic Lateral Sclerosis.
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Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease and a neurodegenerative disorder, affecting the upper and/or lower motor neurons. Notably, it invariably leads to death within a few years of onset. Although most ALS cases are sporadic, familial amyotrophic lateral sclerosis (fALS) forms 10% of the cases. In 1993, the first causative gene (SOD1) of fALS was identified. With rapid advances in genetics, over fifty potentially causative or disease-modifying genes have been found in ALS so far. Accordingly, routine diagnostic tests should encompass the oldest and most frequently mutated ALS genes as well as several new important genetic variants in ALS. Herein, we discuss current literatures on the four newly identified ALS-associated genes (CYLD, S1R, GLT8D1, and KIF5A) and the previously well-known ALS genes including SOD1, TARDBP, FUS, and C9orf72. Moreover, we review the pathogenic implications and disease mechanisms of these genes. Elucidation of the cellular and molecular functions of the mutated genes will bring substantial insights for the development of therapeutic approaches to treat ALS.
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Frontotemporal lobar degeneration (FTLD) is a genetically heterogenous syndrome and has been associated most recently with a hexanucleotide repeat expansion within the C9ORF72 gene. Pathogenic TDP-43 gene (TARDBP) mutations have been identified in amyotrophic lateral sclerosis, but the role of TARDBP mutations in FTLD is more contradictory. To investigate the role of TARDBP mutations in a clinical series of Finnish FTLD patients, we sequenced TARDBP exons 1 to 6 in 77 FTLD patients. No evident pathogenic mutations were found. We identified a novel heterozygous c.876_878delCAG sequence variant in 2 related patients with behavioral variant frontotemporal dementia without amyotrophic lateral sclerosis. The variant is predicted to cause an amino acid deletion of serine at position 292 (p.Ser292del). However, p.Ser292del was also found in 1 healthy middle-aged control. Interestingly, both patients carried the C9ORF72 expansion. Therefore, the TARDBP variant p.Ser292del might be considered a rare polymorphism and the C9ORF72 repeat expansion the actual disease-causing mutation in the family. Our results suggest that TARDBP mutations are a rare cause of FTLD. However, the interaction of several genetic factors needs to be taken into account when investigating neurodegenerative diseases.
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Frontotemporal lobar degeneration
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Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, characterized by progressive degeneration of the upper and lower motor neurons, leading to muscle weakness, hypotrophy, swallowing and respiratory failure. The cause of ALS is not yet fully elucidated, but there are 35 associated genes and 2 gene loci with an unidentified gene. The most common are C9orf72, SOD1, TARDBP and FUS found in approximately 10% of patients. Variants in the C9orf72 gene are the main cause of fALS – 25-40% of cases (and a small percentage of sALS). The goal of the present study was to evaluate the significance of the C9orf72 hexanucleotide repeat expansion in Bulgarian patients with ALS, through the means of in house and triplet repeat-primed PCR assay (TP-PCR). From 171 patients diagnosed with ALS and included in the current study, we have identified the repeat expansion with more than 145 GGGGCC repeats in 7 (4,1%). Short expansions or borderline values (24 to 30 repeats) were not detected. Due to absence of sufficient data, we have established an ALS-focused research for the association of the C9orf72 gene in clinically well-characterized Bulgarian ALS patients. Published data show variable percentage ratios for genetically verified cases (4-40%), which is mainly due to small sample counts and sALS-fALS ratios. Our patients’ group also contains sALS and fALS cases, which explains the low percentage of genetic verification. The obtained results enrich the worldwide database and shed light onto genetically characterized Bulgarian ALS patients. Affected patients and their families can receive adequate medical-genetic consultation and prenatal diagnostic testing.
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